Recent advances in plant genetic engineering have opened new doors to engineer plants to have improved characteristics or traits. These transgenic plants characteristically have recombinant DNA constructs in their genome that have a protein-coding region operably linked to multiple regulatory regions that allow accurate expression of the transgene. A few examples of regulatory elements that help regulate gene expression in transgenic plants are promoters, introns, terminators, enhancers and silencers.
Plant genetic engineering has advanced to introducing multiple traits into commercially important plants, also known as gene stacking. This can be accomplished by multigene transformation, where multiple genes are transferred to create a transgenic plant that might express a complex phenotype, or multiple phenotypes. But it is important to modulate or control the expression of each transgene optimally. The regulatory elements need to be diverse, to avoid introducing into the same transgenic plant repetitive sequences, which have been correlated with undesirable negative effects on transgene expression and stability (Peremarti et al (2010) Plant Mol Biol 73:363-378; Mette et al (1999) EMBO J 18:241-248; Mette et al (2000) EMBO J 19:5194-5201; Mourrain et al (2007) Planta 225:365-379, U.S. Pat. Nos. 7,632,982, 7,491,813, 7,674,950, PCT Application No. PCT/US2009/046968). Therefore it is important to discover and characterize novel regulatory elements that can be used to express heterologous nucleic acids in important crop species. Diverse regulatory regions can be used to control the expression of each transgene optimally.
Regulatory sequences located downstream of protein-coding regions contain signals required for transcription termination and 3′ mRNA processing, and are called terminator sequences. The terminator sequences play a key role in mRNA processing, localization, stability and translation (Proudfoot, N, (2004) Curr Opin Cell Biol 16:272-278; Gilmartin, G. M. (2005) Genes Dev. 19:2517-2521). The 3′ regulatory sequences contained in terminator sequences can affect the level of expression of a gene. Optimal expression of a chimeric gene in plant cells has been found to be dependent on the presence of appropriate 3′ sequences (Ingelbrecht et al. (1989) Plant Cell 1:671-680). Read-through transcription through a leaky terminator of a gene can cause unwanted transcription of one transgene from the promoter of another one. Also, bidirectional, convergent transcription of transgenes in transgenic plants that have leaky transcription termination of the convergent genes can lead to overlapping transcription of the convergent genes. Convergent, overlapping transcription can decrease transgene expression, or generate antisense RNA (Bieri, S. et al (2002) Molecular Breeding 10:107-117). This underlines the importance of discovering novel and efficient transcriptional terminators.